The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we developed a method to freeze-substitute hippocampal cultures and then examine them in thin sections by EM tomography to show individual protein complexes in their natural setting within the PSD. The initial work employing tomography revealed that the core of the PSD is an array of vertically oriented filaments that contain the scaffold protein, PSD-95, in an extended configuration and a polarized orientation, with its N-terminus positioned at the postsynaptic membrane. This finding provided insight into the overall organization of the PSD because scaffolding proteins such as PSD-95 family MAGUK proteins have distinct multiple, diverse binding sites for other proteins arrayed along their length. Thus, the regular arrays of PSD-95, perhaps with other family members, impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. The mechanisms that regulate PSD-95 MAGUK conformations were investigated in collaboration with the William Green laboratory. A new technique, Fluorescent Resonate Energy Transfer (FRET) was applied to measure configurations of PSD-95 molecules in different functional configurations. PSD-95 adopts an extended conformation in PSDs, but remains in closed conformation at non-synaptic sites. In contrast, SAP-97, another MAGUK, adopts an open configuration, but is oriented parallel with the post synaptic membrane. The open conformation of PSD-95 at the PSD is now established as a requirement for it to interact with NMDAR and AMPAR-Stargazin complexes. EM tomography also revealed that the C-terminal ends of the PSD-95 vertical filaments are associated with horizontally oriented filaments. Immunogold labeling identifies one class of horizontal filaments as GKAP, which is a known to bind to the GK domain at the C-terminal end of PSD-95. The emerging structural model of the PSD shows how the PSD-95 matrix can stabilize glutamate receptors forming elaborate molecular complexes, and at the same time allows room for the addition of new receptors at the edges of the PSD. We are continuing this line of work with EM tomography to unravel how the GKAP-Shank and perhaps Homer scaffolding system might contribute to the proper functions of glutamate receptors. Direct identification of the components of the PSD is difficult and we attempted to develop an improved method for identifying the proteins using an expressible probe, miniSOG, confirming that the vertical filaments are PSD-95, however, the staining generated by miniSOG is, so far, too diffuse to localize molecules with better than 20 nm precision, so further fine tuning of this approach is necessary to overcome this problem. The idea that the PSD-95 dependent scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of RNAi knock down of MAGUKs. Recently, we examined the effects of knocking down simultaneously three major MAGUK proteins: PSD-95, PSD-93 and SAP102, and EM tomography revealed significant loss from the central core of the PSD, including NMDA receptor structures, vertical filaments, and AMPA receptors. Electrophysiology measurements by collaborators from the Roger Nicoll laboratory characterizing the effects of the same knock down show significant functional loss of NMDAR and AMAPR type EPSPs at levels compatible with the structural losses. Electron microscopy, also showed depletion of vertical filaments along with AMPAR type structures at the peripheral region of the PSD, and significant reduction in size of NMDAR clusters in the center of the PSD. These structural data indicate that vertical filaments corresponding to MAGUKs anchor AMPARs and are also a factor in organizing NMDARs. Thus, PSD-95 MAGUKs are demonstrated to be the essential organizer of glutamate receptors at the PSD. Continuing on this line of work, we are trying to identify NMDARs more directly in intact hippocampal synapses by combining CRISPR-Cas9 knockout of NMDARs and reconstruction of the postsynaptic density (PSD) with dark field scanning EM tomography. We now have new evidence that the class of transmembrane structures containing larger globular cytoplasmic profile likely contain NMDARs whereas the structures with smaller and flat cytoplasmic profile likely contain AMPARs. Being able to directly identify NMDAR and AMPARs paves the way to visualizing the changes in receptor distributions underlying synaptic plasticity. A new collaboration with the M. Constantine-Patton lab seeks to determine the structure of synapses in a Flailer mouse mutant where many of actin based myosin V motors are inactive. This defect leads to significant reduction in PSD-95 MAGUKs, as well as GKAPs and Shank-Homer molecules in the dendritic spines, and greatly reduced synaptic transmission. So far, we have examined disassociated cultures of flailer mice, and will proceed with serial section EM to compare the structural differences in dendritic spines and PSDs of control and mutant mice. Ultimately, analysis by EM tomography will sort out the actual molecular deficits in this mutant. We have also developed a collaboration with M. DellAcqua, University of Colorado, to study conformations and distribution of A Kinase Anchoring Proteins (AKAPs) at hippocampal synapses. These molecules are membrane associated proteins known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA, PKC) and calcineurin, important for synaptic plasticity (LTP and LTD). This work is showing that there is a conformational change in AKAPs in the PSD, different from that at the extrasynaptic membrane. This distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. In collaboration with the Roger Nicoll lab, we are studying the effects of over expressing constitutively activated CaMKII on synaptic structure and function. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM. Finally, we have developed a new electron microscopic method in collaboration with Richard Leapman using dark field STEM tomography for sections up to 300-400 nm thick to provide detailed reconstructions of whole PSDs. The darkfield imaging, which provides enhanced visualization of the smallest structures at the PSDs provides an opportunity to reconstruct detailed molecular organization of more or less complete PSDs in intact neurons. A new initiative is an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility. Dr. Smith has cultured a primitive animal that is remarkable in that it lacks synapses but shows behavior indicative of neural function. These results are compatible with an early stage in evolving nervous systems, prior to the development of synapses, that utilizes peptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. A cell that senses direction of gravity and mediates behavior accordingly has also been discovered, but what control systems are utilized is not yet clear. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors are expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains.